Ethanol is an important raw material for food and chemical industry and a renewable fuel. At present, cellulose ethanol production technology is under development, and can not meet the requirements of commercialization. The ethanol on sale is mainly produced from corn and sugar cane as the raw materials, and such raw materials are rich in starch and sugar, respectively. In China, in addition to corn, wheat and cassava and other starch-rich raw materials are also used to produce ethanol. It is very important to improve the existing technology and reduce the processing cost of starch raw materials to ethanol.
At present, the ethanol production from starch raw materials needs a number of steps, such as: crushing, liquefaction, saccharification, fermentation, distillation and so on. In these steps, the addition of saccharifying enzymes (ie, glucose amylase, EC 3.2.1.3) is required in the saccharification process for degrading the starch into glucose. The enzyme can be purchased directly from the market. The cost of the saccharification enzymes needed for the production of one ton of ethanol is about 100 rmb. If the fermentative strains possess the function of a glucoamylase, then the amount of glucoamylase can be reduced. Therefore, for reducing the production cost of ethanol, it is necessary to develop Saccharomyces cerevisiae with saccharification function.
From the mid-1980s, glucoamylases from various sources were cloned into Saccharomyces cerevisiae to construct yeast strains with saccharification function.
In 1985, Cetus published a paper on Science and reported that glucamylase from Aspergillus awamori was cloned into Saccharomyces cerevisiae by genetic engineering. The recombinant strain was able to grow on starch as the sole carbon source. In addition to Aspergillus awamori sources, Rhizopus oryzae and Aspergillus niger-derived glucamylases are often used. For increasing the ability to saccharify starch, α-amylase (EC3.2.1.1) was subsequently cloned into Saccharomyces cerevisiae, for saccharifying starch in combination with glucoamylase. Bacillus amyloliquefaciens, Bacillus stearothermophilus, Bacillus licheniformis, Bacillus subtilis, Streptococcus bovis, Debaryomyces occidentalis and barley (Hordeum vulgare) etc. are common sources of α-amylase. It has been reported that the co-expression of α-amylase derived from streptococcus bovis, not from bacillus stearothermophilus with Rhizopus oryzae-derived glucoamylase can induce the saccharification and fermentation of the recombinant strain with a raw starch as a raw material. The reason is that the bacillus stearothermophilus-derived α-amylase does not have a starch-binding domain, and its co-expression with Rhizopus oryzae-derived glucoamylase can only cause the saccharification and fermentation of the recombinant strain with a soluble starch as a raw material. In addition, debranching enzymes (E.C.3.2.1.33 pullulanase and isoamylase) and maltose transporters were also cloned into Saccharomyces cerevisiae to enhance the saccharification and fermentation of the starch.
Recently, some research teams use surface display technology to construct Saccharomyces diastaticus, while others are committed to construct Saccharomyces diastaticus using the secretory expression technology. However, the current research is mostly based on basic research, and it is difficult to meet the requirements of industrial applications. For example, the used host is a laboratory strain with auxotrophic phenotype, which makes the recombinant bacteria not strong enough, and brings about some other issues, such as low growth rate and low fermentation performance; and a 2 μm of plasmid expression vector is usually used, the benefit of which is that it is a multi-copy; and the disadvantage of which is that, under the conditions of the non-selective pressure, it is easy to lose.
Therefore, it is still necessary to develop a Saccharomyces cerevisiae with efficient saccharification function qualified with the requirements of industrial application.